Erratum Werner syndrome protein participates in a complex with RAD51, RAD54, RAD54B and ATR in response to ICL-induced replication arrest Marit Otterlei, Per Bruheim, Byungchan Ahn, Wendy Bussen, Parimal Karmakar, Kathy Baynton and Vilhelm A. Bohr Journal of Cell Science 119, 5215 (2006) doi:10.1242/jcs.03359 There was an error in the first e-press version of the article published in J. Cell Sci. 119, 5137-5146. The first e-press version of this article gave the page range as 5114-5123, whereas it should have been 5137-5146. We apologise for this mistake. Research Article 5137 Werner syndrome protein participates in a complex with RAD51, RAD54, RAD54B and ATR in response to ICL-induced replication arrest Marit Otterlei1,2,*, Per Bruheim1,3,§, Byungchan Ahn1,4,§, Wendy Bussen5, Parimal Karmakar1,6, Kathy Baynton7 and Vilhelm A. Bohr1 1Laboratory of Molecular Gerontology, National Institute on Aging, NIH, 5600 Nathan Shock Dr., Baltimore, MD 21224, USA 2Department of Cancer Research and Molecular Medicine, Laboratory Centre, Faculty of Medicine, Norwegian University of Science and Technology, Erling Skjalgsons gt. 1, N-7006 Trondheim, Norway 3Department of Biotechnology, Norwegian University of Science and Technology, N-7491 Trondheim, Norway 4Department of Life Sciences, University of Ulsan, Ulsan 680-749, Korea 5Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, 333 Cedar St, SHM-C130, New Haven, CT 06515, USA 6Department of Life Science and Biotechnology, Jadavpur University, Kolkata-700 032, WB, India 7Research Center, Ste. Justine’s Hospital, 3175 Cote Ste. Catherine, Montreal, Quebec H3T 1C5, Canada *Author for correspondence (e-mail: [email protected]) §These authors contributed equally to this work Accepted 5 October 2006 Journal of Cell Science 119, 5137-5146 Published by The Company of Biologists 2006 doi:10.1242/jcs.03291 Summary Werner syndrome (WS) is a rare genetic disorder resonance energy transfer (FRET) and immune- characterized by genomic instability caused by defects in precipitation experiments to demonstrate that WRN the WRN gene encoding a member of the human RecQ participates in a multiprotein complex including RAD51, helicase family. RecQ helicases are involved in several DNA RAD54, RAD54B and ATR in cells where replication has metabolic pathways including homologous recombination been arrested by ICL. We verify the WRN-RAD51 and (HR) processes during repair of stalled replication forks. WRN-RAD54B direct interaction in vitro. Our data Following introduction of interstrand DNA crosslinks support a role for WRN also in the recombination step of (ICL), WRN relocated from nucleoli to arrested replication ICL repair. forks in the nucleoplasm where it interacted with the HR protein RAD52. In this study, we use fluorescence Key words: WRN, ICL, RAD51, RAD54B, ATR, DNA repair Journal of Cell Science Introduction The RecQ helicases display 3Ј to 5Ј unwinding directionality Individuals with Werner syndrome (WS) are afflicted with a on DNA on a variety of structures including recombination genomic instability disorder derived from defects in the WRN intermediates such as Holliday junctions (HJ) (Constantinou et gene encoding a member of the RecQ DNA helicase family al., 2000). The WRN protein is unique among human RecQ (Martin, 1978; Yu et al., 1996). RecQ helicases are highly helicases in that it also contains a 3Ј to 5Ј exonuclease activity conserved from bacteria to human; however, bacteria possess a (Huang et al., 1998). How the WRN helicase and exonuclease single RecQ enzyme, whereas in humans, five RecQ members function to resolve DNA structures in vivo is currently unclear; have been identified so far (Hickson, 2003). Deficiencies in three however, cooperativity between the two activities has been of the human RecQ helicases, WRN (Martin et al., 1970), BLM suggested to occur in some situations (Opresko et al., 2003; and RECQL4, are responsible for Werner syndrome (WS), Opresko et al., 2001; Opresko et al., 2004). Bloom syndrome (BS), and Rothmund-Thomson syndrome WRN interacts physically and functionally with a number of (RTS), respectively. All three disorders are characterized by proteins involved in different DNA metabolic pathways decreased genomic stability leading to increased cancer including replication protein A (RPA) (Brosh et al., 1999), incidence (Hickson, 2003). WS is further classified as a proliferating cell nuclear antigen (PCNA) (Lebel et al., 1999), segmental progeroid syndrome since patients display the early polymerase ␦ (Kamath-Loeb et al., 2000), flap endonuclease 1 onset of many age-associated pathologies, such as (FEN-1) (Brosh et al., 2001), RAD52 (Baynton et al., 2003), arteriosclerosis, cataracts, diabetes mellitus type II, greying the MRN complex through binding to NBS1 (Cheng et al., and loss of the hair, wrinkling of the skin, osteoporosis and 2004), the Ku heterodimer (Cooper et al., 2000; Hsu et al., increased predisposition to cancer development, primarily of 2000), and TRF2 (Opresko et al., 2002). Thus, WRN may be mesenchymal origin (Hickson, 2003; Opresko et al., 2003). Cells a multi-tasking enzyme that functions in several genome from WS patients display marked chromosomal rearrangements maintenance pathways, possibly by co-ordinating their and deletions (Fukuchi et al., 1989; Grigorova et al., 2000; Salk, progression. 1982) and a shortened life span in culture (Martin et al., 1970). One process in which WRN function has been strongly The molecular basis of the premature senescence and the implicated is the recovery of arrested replication forks genomic instability in WS cultured cells is not yet clear. (Baynton et al., 2003; Sakamoto et al., 2001). Replication fork 5138 Journal of Cell Science 119 (24) arrest and collapse may lead to double-strand breaks (DSB) and WRN was recently found to be required for ICL repair in that are repaired in mammalian cells by either non homologous an in vitro assay and thus suggested to be a candidate helicase end joining (NHEJ) or homologous recombination (HR) (for involved in the early steps in processing ICL (Zhang et al., reviews, see Lieber et al., 2003; West, 2003). The essentially 2005). In the early pre-synapsis step of HR, a single-stranded error-free HR repair pathway is currently viewed as the major DNA (ssDNA) tail is converted to a nucleoprotein filament mechanism for re-establishing stalled replication forks during consisting of RPA, RAD51, RAD52, RAD54 and possibly S-phase in human somatic cells. The current model for the RAD54B (Alexeev et al., 2003; Tan et al., 2003; Tanaka et al., repair and restart of an arrested replication fork involves a 2000). The formation of WRN and RAD52 complexes at structure called a ‘half chicken foot’ or a ‘chicken foot’ arrested replication forks induced by ICL, together with the resembling a Holliday junction that can be resolved by observed stimulation of RAD52 strand annealing activity by recombination (reviewed in Helleday, 2003). Studies in WRN in vitro, suggests that WRN may function both during Escherichia coli indicate that the ‘chicken foot’ can be early steps of processing ICL (Zhang et al., 2005; Zhang et al., processed by the DNA helicase activity of RecQ together with 2003) and during pre-synapsis in later steps of ICL repair. the 5Ј to 3Ј exonuclease function of RecJ prior to replication To evaluate further the potential role of WRN in ICL restart (Courcelle et al., 2003). Interestingly, recent data repair/recovery processes, we examined whether WRN have shown that WRN helicase unwinds the chicken-foot interacts with other members of the RAD52 epistasis group intermediate and stimulates FEN-1 to cleave the unwound following replication arrest. We report here that WRN re- product in a structure-dependent manner (Sharma et al., 2004). localizes to a complex (or complexes) composed of RAD54, However, the precise steps and proteins involved in this process RAD51, RAD54B and ATR in response to replication arrest are still unclear (reviewed in Helleday, 2003; Hickson, 2003; induced by treatment of cells with MMC. In vitro biochemical West, 2003) and under intense investigation. analyzes reveal that WRN associates directly with RAD51 and RecQ helicases and RAD HR proteins have also been RAD54B. Based on our data we suggest a role for WRN in the documented to collaborate in DNA maintenance pathways resolution of stalled replication forks arrested by ICL, both as distinct from stalled replication fork recovery. The the candidate helicase during the early steps of processing ICL Saccharomyces cerevisiae RecQ helicase homolog, Sgs1p, as suggested by Zhang et al. (Zhang et al., 2005) and during functions together with RAD52 epistasis group members in a the later pre-synapsis step of homologous recombination. telomerase-independent alternative lengthening of telomeres (ALT) pathway (Le et al., 1999; Teng and Zakian, 1999). ALT Results pathways have also been detected in a number of immortalized Nuclear localization dynamics of WRN, RAD51, RAD54 human cell lines lacking telomerase (Bryan et al., 1997; Colgin and RAD54B in untreated cells and Reddel, 1999), and ALT cells contain ALT-associated Previous fluorescence resonance energy transfer (FRET) data PML bodies consisting of promyelocytic leukaemia protein demonstrated that prolonged exposure (16-24 hours) of HeLa (PML), telomeric DNA, the telomere binding proteins TRF1 cells to MMC leads to the re-localization of RAD52 and WRN and TRF2, the RAD homologous recombination (RAD-HR) to sites of arrested replication where they physically interact Journal of Cell Science proteins RAD50, RAD51, RAD52, MRE11, NBS1, ATR, and (Baynton et al., 2003). To study intracellular localization and the BLM and WRN helicases (Barr et al., 2003; Henson et al., re-localization dynamics as well as to identify potential protein- 2002). WRN mutations have been associated with increased protein interactions between WRN and other HR components telomere dysfunction in cells cultured from later generation upon replication fork blockage using fluorescence microscopy mice lacking the telomerase RNA component, Terc (Chang et and FRET analysis, we cloned human RAD51, RAD54 and al., 2004).